Identification of novel pathogenic variants of Calpain-3 gene in limb girdle muscular dystrophy R1

Abstract

Background: Limb Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disease caused by mutations in the calpain-3 (CAPN3) gene. As clinical and pathological features may overlap with other types of LGMD, therefore definite molecular diagnosis is required to understand the progression of this debilitating disease. This study aims to identify novel variants of CAPN3 gene in LGMDR1 patients.

Results: Thirty-four patients with clinical and histopathological features suggestive of LGMD were studied. The muscle biopsy samples were evaluated using Enzyme histochemistry, Immunohistochemistry, followed by Western Blotting and Sanger sequencing. Out of 34 LGMD cases, 13 patients were diagnosed as LGMDR1 by immunoblot analysis, demonstrating reduced or absent calpain-3 protein as compared to controls. Variants of CAPN3 gene were also found and pathogenicity was predicted using in-silico prediction tools. The CAPN3 gene variants found in this study, included, two missense variants [CAPN3: c.1189T > C, CAPN3: c.2338G > C], one insertion-deletion [c.1688delinsTC], one splice site variant [c.2051-1G > T], and one nonsense variant [c.1939G > T; p.Glu647Ter].

Conclusions: We confirmed 6 patients as LGMDR1 (with CAPN3 variants) from our cohort and calpain-3 protein expression was significantly reduced by immunoblot analysis as compared to control. Besides the previously known variants, our study found two novel variants in CAPN3 gene by Sanger sequencing-based approach indicating that genetic variants in LGMDR1 patients may help to understand the etiology of the disease and future prognostication.

Keywords: Calpain-3; Immunohistochemistry; LGMDR1; Sanger sequencing; Western blotting.

Fig. 1

Photomicrograph showing variation in muscle fibre size, lobulated muscle fibres (black arrow), atrophic fibres (blue arrow) and internalization of nuclei (a: H and E x 20X, b: H and E x 40X)

Fig. 2

Enzyme histochemistry showing lobulated fibres a: NADH-TR, b: COX, c: SDH stains and d: combined COX-SDH for highlighting COX-deficient fibre (arrow) x 40X

Fig. 3

Immunohistochemistry using antibodies against Dystrophin, Sarcoglycan-alpha, Sarcoglycan-beta, Sarcoglycan-gamma and Dysferlin showing normal expression in myofibres (a: Dystrophin x 20X, b: Sarcoglycan-alpha x 40X, c: Sarcoglycan-beta x 20X, d: Sarcoglycan-gamma x 40X, e: Dysferlin x 40X)

Fig. 4

Immunoblot analysis of calpain-3 protein in LGMD patients (a) Decreased band intensity of calpain-3 protein at 94 kDa in patients as compared to control samples [The full image of membrane was not available. The membrane was cut and hybridised one part with anti-calpain-3 antibody and another part with anti-GAPDH antibody]; (b) Calpain-3 protein expression was significantly reduced in patients (13 LGMDR1 patients) as compared to control [C- Control; P- Patients 1–6]

Fig. 5

Homozygous missense variant in exon 9 of CAPN3 gene (Patient 1) [(NM_000070.3 (CAPN3): c.1189T > C; NP_000061.1: p.Phe397Leu (Novel variant)] [a - control, b - patient]

Fig. 6

Homozygous missense variant in exon 22 of CAPN3 gene (Patient 2) [NM_000070.3 (CAPN3): c.2338G > C; NP_000061.1: p.Asp780His (rs778768583)] [a - control, b - patient]

Fig. 7

Homozygous missense variant in exon 22 of CAPN3 gene (Patient 5) [NM_000070.3 (CAPN3): c.2338G > C; NP_000061.1: p.Asp780His (rs778768583)] [a - control, b– patient]

Fig. 8

Heterozygous missense variant in exon 22 of CAPN3 gene (Patient 3) [NM_000070.3 (CAPN3): c.2338G > C; NP_000061.1: p.Asp780His (rs778768583)] [a - control, b– patient, c- patient’s father]

Fig. 9

Homozygous insertion-deletion in exon 11 of CAPN3 gene (Patient 4) [NM_000070.3 (CAPN3): c.1688delinsTC; NP_000061.1: p.Arg490Leufs*87 (Novel variant)] [a - control, b - patient]

Fig. 10

Splice site changes in intron 18 of CAPN3 gene (Patient 3) [NM_000070.3 (CAPN3): (c.2051-1G > T) (rs886042108)] [a - control, b– patient, c - patient’s mother ]

Fig. 11

Homozygous nonsense variant in exon 17 of CAPN3 gene (Patient 6) [NM_000070.3 (CAPN3): c.1939G > T; NP_000061.1: p.Glu647Ter (rs863224960)] [a - control, b - patient]